Input and output¶

labelZmws¶

The labelZmws command takes an input.fofn representing a set of bas.h5 files to operate on. Additionally, it takes a barcode.fasta file. Depending on scoreMode, the FASTA file will be processed in different ways. Specifically, in paired mode, each two consecutive barcodes in the file are considered a set.

The parameters, adapterSidePad and insertSidePad represents how many bases should be considered on each side of the putative barcode. These parameters are constrained such that: |adapterSidePad| + |insertSidePad| + |barcode| < 65.

Users have the option to specify a different output location for the various outputs. Specifically, for each bas.h5 file in input.fofn, a bc.h5 (barcode hdf5) file is generated. These files are listed in the file outFofn which is typically just called barcode.fofn. See below for a description of the barcode hdf5 file.

labelAlignments¶

The labelAlignments command takes as input a barcode.fofn computed from a call to labelZMWs and a cmp.h5 file where the barcode information is written to. See below for a description of the cmp.h5 file additions.

emitFastqs¶

The emitFastqs command takes as input both an input.fofn for the bas.h5 files as well as a barcode.fofn from a call to labelZmws. The optional parameter outDir dictates where the files will be written. For each detected barcode, a fast[a|q] file will be emitted with all of the reads for that barcode. The trim parameter dictates how much of the read should be trimmed off. The default parameter for trim is the length of the barcode (which is stored in the barcode hdf5 files). At the moment, all barcodes in the barcode FASTA file must be the same length, therefore only a constant trim value is supported. In practice, one can aggressively trim in order to ensure that extra bases aren't left on the ends of reads. Finally, the subreads parameter dictates whether subreads or CCS reads should be returned with the default being the appropriate reads according to the input file type, either CCS or subreads. This parameter is only inspected if the input.fofn contains both CCS and subread data, if the input.fofn contains only subread or CCS data then that is returned irrespective of the state of the the subreads parameter and a warning is issued.

consensus¶

The emitFastqs command takes as input both an input.fofn for the bas.h5 files as well as a barcode.fofn from a call to labelZmws. The results are a FASTA file with an entry for each barcode containing the consensus amplicon sequence. This mode utilizes Quiver and pbdagcon to compute consensus.

In cases where the amplicon is fewer than 2.5k bases, using CCS data is quite helpful. The --ccsFofn allows one to pass directly the ccs files. In many cases, both the CCS and raw basecalls are in the same file so you can check by passing the same parameter to input.fofn as to ccsFofn.

Dependencies¶

Barcode HDF5 File¶

/BarcodeCalls/best - (nZMWs, 6)[32-bit integer] dataset with the following columns:

Additionally, the best dataset has the following attributes:

The two barcodeIdx1 and barcodeIdx2 columns are indices into barcodes attribute. The scoreMode is scoring mode used to align the barcodes. The barcodes attribute correspond to the barcode.fasta sequence names.

Additionally, in some circumstances, it is useful to retain the entire history of the scoring, i.e., each barcode scored to each adapter across all ZMWs. In oder to retain this information, one must call:

In this mode, the resultant HDF5 file will have an additional dataset under the BarcodeCalls group, named: all. This dataset has the following format:

/BarcodeCalls/all - (nbarcodes * nadapters[zmw_i], 4) forall i in 1 ... nZMWs

The adapterIdx is the index of the adapter along the molecule, i.e., adapterIdx 1 is the first adapter scored.

Additions to the compare HDF5 (cmp.h5) File¶

/BarcodeInfo/



ID   (nBarcodeLabels + 1, 1)[32-bit integer]
Name (nBarcodeLabels + 1, 1)[variable length string]

In addition to the /BarcodeInfo/ group, the key dataset which assigns alignments to barcodes is located at:

/AlnInfo/Barcode (nAlignments, 3)[32-bit integer] with the following columns:

Here index refers to the index into the Name vector, score corresponds to the sum of the scores for the barcodes, and finally, count refers to the number of adapters found in the molecule.